The relevance of bladder urothelial cells (BUG) to human disease has traditionally been understood only in terms of urothelial cell carcinoma. However, there is exciting new literature highlighting the role of altered BUG structure and function in non-neoplastic states. Two highly prevalent yet poorly understood conditions, interstitial cystitis (IC) and overactive bladder (OAB) may both be associated with pathogenic changes in the bladder urothelium. Both of these conditions are characterized by hypersensory symptoms such as urinary urgency, frequency, and bladder pain. We propose to study the structure and function of the bladder urothelium as it relates to both normal and hypersensory conditions of IC and OAB. Recent scanning electron microscopy of the lumenal side of the bladder urothelium show distinct changes between IC and control apical umbrella cells (AUC). These changes could be due to abnormalities in uroplakins expression. Uroplakins are unique proteins expressed only by the AUC in the urothelium and play a role in AUC membrane structure and function. Human IC BUC grown in culture also had abnormalities including augmented purinergic signaling, altered expression of certain growth factors (HB-EGF, EGF and APF), and decreased potassium conductance (mediated by ion channels Kir2.1 and BK) leading to depolarization. The discovery of muscarinic M2 receptors on the bladder urothelium provides another mechanism of action in the use of antimuscarinics in treatment of OAB. This grant will test the following 4 hypotheses: 1. AUC from patients with bladder hypersensory conditions have structural abnormalities related to uroplakins. 2. The bladder urothelium from patients with bladder hypersensory conditions have altered protein expressions of tight junction proteins (ZO-1, occludin), Kir2.1, BK, M2, HB-EGF, EGF and APF. 3. The conductance of ion channel Kir2.1 is decreased in IC BUC. This can be reversed with HB-EGF treatment. Normal BUC can be made to have decreased Kir2.1 conductance with APF treatment. 4. The conductance of the BK channel is regulated by M2 receptors in human BUC. There is a decreased outward potassium current in OAB and IC BUC which can be reversed by hSIo cDNA transfection. By accomplishing these aims, we hope to develop an understanding of the pathogenesis of hypersensory bladder conditions which ultimately can lead to targeted treatment options.